The Journal of Neuroscience, March 15, 1999, 19(6):2090–2101

Specification of Distinct Neural Pathways: Roles of the Eph Family Receptor EphB1 and Ligand Ephrin-B2

Yong Yue,1 David A. J. Widmer,2 Alycia K. Halladay,2 Douglas Pat Cerretti,3 George C. Wagner,2 Jean-Luc Dreyer,4 and Renping Zhou1 1Laboratory for Cancer Research, College of Pharmacy, and 2Department of Psychology, Rutgers University, Piscataway, New Jersey 08855, 3Immunex Corporation, Seattle, Washington 98101, and 4Department of Biochemistry, University of Fribourg, 1700 Fribourg, Switzerland

Dopaminergic neurons in the and ventral teg- cell loss of substantia nigra, but not ventral tegmental, dopa- mental area project to the caudate and nucleus minergic neurons. These studies suggest that the ligand–recep- accumbens/, respectively, constituting meso- tor pair may contribute to the establishment of distinct neural striatal and mesolimbic pathways. The molecular signals that pathways by selectively inhibiting the neurite outgrowth and confer target specificity of different dopaminergic neurons are cell survival of mistargeted neurons. In addition, we show that not known. We now report that EphB1 and ephrin-B2, a recep- ephrin-B2 expression is upregulated by cocaine and amphet- tor and ligand of the Eph family, are candidate guidance mol- amine in adult mice, suggesting that ephrin-B2/EphB1 interac- ecules for the development of these distinct pathways. EphB1 tion may play a role in drug-induced plasticity in adults as well. and ephrin-B2 are expressed in complementary patterns in the dopaminergic neurons and their targets, and the li- Key words: axonal guidance; dopaminergic pathways; Eph gand specifically inhibits the growth of neurites and induces the family receptors; ephrins; drug ; plasticity

The midbrain dopaminergic neurons are located primarily in two receptors (Zhou, 1998). This family is unique among tyrosine adjacent regions, the more lateral substantia nigra and the more kinase receptors in that all of the ligands are membrane-anchored medial , and project to the forebrain, form- and in that the association is required for activity (Davis et al., ing several distinct pathways (Ungerstedt, 1971; Moore and 1994). The soluble ligands lack biological activity and can func- Bloom, 1978; Lindvall and Bjorklund, 1983; Di Chiara et al., tion as antagonists (Gao et al., 1996). The requirement of mem- 1992; Heimer et al., 1995). Ventral tegmental dopaminergic neu- brane anchorage makes the Eph family ligands uniquely qualified rons send their to the ventromedial (which in- as local guidance cues for axonal targeting. cludes and olfactory tubercle), constituting In vivo and in vitro observations indicate that the Eph family the , and to the cortex () ligands and receptors play key roles in the specification of topo- (Simon et al., 1976, 1979; Nauta et al., 1978; Beckstead et al., graphic projections. In the retinotectal and hippocamposeptal 1979). In contrast, substantia nigra dopaminergic neurons selec- systems, Eph family ligands and receptors are expressed in pro- tively project to dorsolateral striatum (caudate putamen) in an jecting and target fields in opposing gradients (Cheng et al., 1995; orderly medial-to-lateral arrangement, forming the nigrostriatal Drescher et al., 1995; Gao et al., 1996; Zhang et al., 1996). pathway (Carter and Fibiger, 1977; Fallon et al., 1978; Nauta et Consistent with the expression patterns, ligand–receptor interac- al., 1978; Beckstead et al., 1979; van der Kooy, 1979). Although tions repel and/or inhibit the growth of receptor-positive axons, these pathways have been identified for nearly three decades, the thus contributing to the specification of topographic projection underlying molecular mechanisms for the development of these maps (Drescher et al., 1995; Gao et al., 1996, 1998; Nakamoto et distinct pathways are still unknown. al., 1996). The Eph family receptor tyrosine kinases and their ligands To examine the roles of the Eph family ligands and receptors in have been implicated in guiding axons during the development of the development of the ascending midbrain dopaminergic path- the nervous system (for review, see Harris and Holt, 1995; ways, we investigated the developmental expression of these guid- Tessier-Lavigne, 1995; Friedman and O’Leary, 1996; Orioli and ance molecules in the dopaminergic neurons and their projection Klein, 1997; Pasquale, 1997; Flanagan and Vanderhaeghen, 1998; targets, including the caudate putamen, nucleus accumbens, and Zhou, 1998). The Eph family is the largest known group of olfactory tubercle. We further examined the biological effects of receptor tyrosine kinases, including at least eight ligands and 14 the Eph ligands on dopaminergic neurons and their regulation by cocaine and in vivo. Our studies indicate that the Received July 16, 1998; revised Nov. 23, 1998; accepted Dec. 22, 1998. Eph family receptors and ligands may play important roles in This research was funded by the National Institute on Drug Abuse (Grant regulating the formation of distinct dopaminergic pathways and 1RO1DA11480-01) and by an Exploratory Research grant from the National Insti- may contribute to drug-induced neural plasticity. tute of Environmental and Occupational Health Sciences. We thank W. Schultz for the critical reading of this manuscript. MATERIALS AND METHODS Correspondence should be addressed to Dr. Renping Zhou, Laboratory for Cancer Research, Department of Chemical Biology, College of Pharmacy, Rutgers Animals and preparation of tissue sections. CD-1 mice from embryonic day University, Piscataway, NJ 08855. (E) 14 to postnatal day (P) 60 (adult) were used in in situ hybridization Copyright © 1999 Society for Neuroscience 0270-6474/99/192090-12$05.00/0 experiments. The day of vaginal plug occurrence was defined as E1 and Yueetal.• Specification of Dopaminergic Pathways by Ephrins J. Neurosci., March 15, 1999, 19(6):2090–2101 2091

Table 1. Expression of Eph family receptors in the P7 midbrain neurons

Eph receptor Midbrain region EphA3 EphA4 EphA5 EphA6 EphA7 EphA8 EphB1 EphB2 EphB3 SN Ϫ Ϫ ϩϩ Ϫ Ϫ Ϫ ϩϩϩ Ϫ Ϫ VTA ϪϪϩϩϪϪϪϮϪϪ

Ϫ, Expression below detection; Ϯ, low expression; ϩϩ, moderate expression; ϩϩϩ, strong expression.

the day of birth as P1. At least three animals were investigated at each staining with polyclonal anti-ephrin-B2 antibody (Santa Cruz, CA). Pos- age group. Whole embryos and of postnatal mice were dissected itive stainings were found in the ephrin-B2-transfected cells. In contrast, under carbon dioxide anesthesia and frozen on dry ice. Coronal and no significant staining was observed in parental or vector-transfected sagittal sections of 16 ␮m thickness were cut on a cryostat at Ϫ23°C and NIH-3T3 cells. mounted on slides coated with 2% triethoxy-3-aminopropyl saline (Sig- Neuron culture and staining. The substantia nigra and ventral tegmental Ϫ ma, St. Louis, MO). Then the slides were stored at 80°C before use. area of E18 rat embryos were dissected from the lateral two-thirds and 35 In situ hybridization. mRNA expression was detected with either [ S]- the medial one-third of the ventral mesencephalon, respectively, under a labeled in vitro transcribed riboprobes or end-labeled oligonucleotide microscope in PBS. The precise positions of substantia nigra and ventral probes. In situ hybridization was performed as described (Zhang et al., tegmental area were determined by tyrosine hydroxylase (TH) immuno- 1997). The optimal exposure time of hybridized sections to autoradio- histochemical staining of E18 rat midbrain sections. Neurons then were graphic emulsion was determined by exposing the sections to x-ray film dissociated and cocultured with monolayer control NIH-3T3 cells or for various time periods before dipping. For quantitative in situ hybrid- ephrin-B2-expressing fibroblasts in DMEM supplemented with fetal ization, complete sets of sections from different age or treatment ␮ groups were analyzed in the same experiment with the same probe. bovine serum (10%), penicillin (50 U/ml), and streptomycin (50 g/ml). Different samples were compared only within the same experiment. Neurons were cultured for various times and stained with a monoclonal Developmental expression of nine Eph family tyrosine kinase recep- anti-TH antibody (Chemicon, Temecula, CA). The number of neurons tors was investigated via in situ hybridization. Only EphB1 showed and the lengths of neurites were measured with a Zeiss Telaval 31 specific differential expression in the midbrain dopaminergic neurons. microscope (Oberkochen, Germany). For quantitation of each sample, 20 EphB1 mRNA was detected with a riboprobe transcribed in vitro from a random fields were chosen from top to bottom and from the left to right 2.4 kb mouse EphB1 cDNA containing most of the coding region cloned sides of culture wells to represent the entire culture. in a pBluescript SK plasmid. Antisense riboprobe was synthesized with Drug treatment. From 30 to 180 mg/kg body weight of cocaine or T7 RNA polymerase for hybridization, and a sense control probe was D-amphetamine was injected subcutaneously in the neck of mice in four generated with T3 RNA polymerase. The synthesized riboprobes were divided doses over 6 hr. The mice were killed 6 hr or 1, 4, 7, or 14 d after hydrolyzed to smaller fragments (ϳ0.2 kb) by 0.2 M bicarbonate at 60°C treatment, and the expression of ephrin-B2 in the striatum was analyzed for 41.6 min before hybridization. with in situ hybridization. To examine the expression of the ligands of EphB1, we performed in situ hybridization analyses with probes of all three ephrin-B ligands. Only ephrin-B2 showed differential expression in the dopaminergic target RESULTS fields. Ephrin-B2 mRNA was detected with a riboprobe made froma1kb human ephrin-B2 cDNA containing the full coding region in pBluescript Expression of Eph family receptor EphB1 in the Ϫ SK . Human ephrin-B2 has a 93% homology with mouse ephrin-B2 in midbrain dopaminergic neurons the nucleotide level (Bergemann et al., 1995; Cerretti et al., 1995). To examine the roles of the Eph family receptors in the develop- Quantitative analysis of EphB1 hybridization signals in the midbrain dopamine system and of ephrin-B2 signals in the striatum was performed ment of the midbrain dopaminergic system, we investigated the with ImagePro 1.3 image analysis software. To avoid bias introduced by expression of nine Eph family receptors that have been shown to the variations of cell density in different regions, we quantitated only be transcribed in the nervous system (for review, see Zhou, 1998), areas with comparable cell density for silver grain density, expressed as using in situ hybridization in P7 mouse brain. These studies a percentage of the area covered by silver grains (area fraction analysis). For each experimental group, hybridization and measurements were showed that seven Eph receptors, EphA3, EphA4, EphA6, done on three animals and on both sides of the regions of interest over EphA7, EphA8, EphB2, and EphB3, were not expressed at sig- multiple sections covering the entire anterior–posterior extension. nificant levels in the midbrain dopaminergic neurons (Table 1). 6-Hydroxydopamine treatment. Five late-pregnant CD-1 mice obtained One receptor, EphA5, was transcribed at moderate levels in both from Charles River Laboratories (Wilmington, MA) were housed indi- vidually in pan cages until pups were born. On day 3 of life, pups in all the substantia nigra and the ventral tegmental area, consistent groups were given a subcutaneous injection of 25 mg/kg pargyline plus 25 with a previous report (Maisonpierre et al., 1993) (Table 1). Only mg/kg imipramine (Sigma). Under surgical anesthesia 60 min later the the remaining receptor, EphB1, was transcribed differentially in ␮ pups were injected in the left ventricle with 50 or 100 gof the ventral mesencephalon (Fig. 1). EphB1 expression was re- 6-hydroxydopamine base dissolved in 5 ␮l of 0.1% ascorbic acid. On day 6 the procedure was repeated with the same dose in the right ventricle. stricted primarily to the substantia nigra. Few transcripts were Ventricle position was determined as Ϯ 1.0 mm medial/lateral from detected in the ventral tegmental area. Although thionine stain- Ϫ bregma and 2.5 mm ventral to the skull surface. Pups then were ing showed a comparable cell density (Fig. 1A), the hybridization allowed to recover for 10 d, at which time they were killed. For EphB1 in ϳ situ hybridization analysis the whole brain was dissected and frozen on signals were significantly higher ( 4.8-fold) in the substantia dry ice. For neurochemical analysis, separate animals were killed, and the nigra than in the ventral tegmental area (Figs. 1B, 2). The striatum was dissected and frozen in liquid nitrogen until assay. difference in EphB1 expression was not attributable to the lack of Levels of dopamine and the metabolites dihydroxyphenylacetic acid dopaminergic neurons in the ventral tegmental area, because TH (DOPAC) and homovanillic acid (HVA) were determined by using HPLC, as described previously (Halladay et al., 1998). immunohistochemical staining using neighboring serial sections Construction of ephrin-B2-expressing cell line. Human full-length showed that the enzyme was found in both the substantia nigra ephrin-B2 was cloned into a retroviral vector pLIG, which contains a and the ventral tegmental area (Fig. 1C). ␤ -galactosidase gene fused to an aminoglycoside phosphotransferase for To demonstrate further that the Eph receptor was expressed in G418 resistance (Lillian, 1996). Then the construct was transfected into National Institutes of Health-3T3 (NIH-3T3) cells. G418-resistant colo- the dopaminergic neurons, we examined the levels of EphB1 nies were screened for ephrin-B2 expression, using immunocytochemical expression in neonatal mice treated with 6-hydroxydopamine, 2092 J. Neurosci., March 15, 1999, 19(6):2090–2101 Yue et al. • Specification of Dopaminergic Pathways by Ephrins

Figure 1. Complementary expression of the Eph family receptor EphB1 and ligand ephrin-B2 in the ascending mesencephalic dopamine systems. A, B, Bright- and dark-field photomicrographs, respectively, of a coronal section of a P2 mouse brain through the ventral mesencephalon hybridized with an antisense EphB1 probe. Hybridized sections were counterstained with thionine to identify the histological patterns. C, Photomicrograph of a neighboring section stained with anti-tyrosine hydroxylase (TH) immunocytochemistry to reveal the substantia nigra and ventral tegmental area. Note that TH staining was observed in both the substantia nigra and the ventral tegmental area, indicating the presence of dopaminergic neurons in both areas. D, E, Bright- and dark-field photomicrographs, respectively, of a P7 coronal mouse brain section through the striatum hybridized with an antisense ephrin-B2 riboprobe and counterstained with thionine. The circled areas are quantitated in Figure 2. ac, Anterior commissure; cc, corpus callosum; LCP, lateral caudate putamen; LS, lateral septum; LV, lateral ventricle; MCP, medial caudate putamen; NA, nucleus accumbens; OT, olfactory tubercle; PC, piriform cortex; SN, substantia nigra; VTA, ventral tegmental area. Scale bars, 0.46 mm.

which selectively destroys dopamine neurons (Stodgell et al., this study, and persists through adult, although higher levels were 1998). The extent of lesion was confirmed by significant decreases found from E18 to P7 (Table 2). in the levels of dopamine and its metabolites, DOPAC and HVA, in the striatum (Fig. 3A). In situ hybridization analysis showed Expression of Eph family ligand ephrin-B2 in that the decrease in the levels of dopamine and its metabolites the striatum was paralleled by a decrease in EphB1 expression levels in the EphB1-positive substantia nigra neurons are known to project to substantia nigra (Fig. 3B). Thus, EphB1 mRNA is transcribed the caudate putamen, but not to the nucleus accumbens and selectively in the substantia nigra dopaminergic neurons. The olfactory tubercle. In contrast, the EphB1-negative ventral teg- expression was observed in E18, the earliest stage examined in mental area dopaminergic neurons project primarily to the nu- Yueetal.• Specification of Dopaminergic Pathways by Ephrins J. Neurosci., March 15, 1999, 19(6):2090–2101 2093

Figure 2. Quantitation of the expression of EphB1 in the midbrain and ephrin-B2 in the striatum. The areas quantitated are indicated in Figure 1. The data presented are the averages of expression levels from three different animals (ϮSEM; *p Ͻ 0.02; ANOVA). LCP, Lateral caudate putamen; MCP, medial caudate putamen; NA, nucleus accumbens; OT, olfactory tubercle; SN, substantia nigra; VTA, ventral tegmental area. cleus accumbens and olfactory tubercle. To test the possibility that the receptor EphB1 and its ligands contribute to target specificity of the midbrain dopaminergic neurons, we examined the expression patterns of all of the ephrin-B subclass ligands, ephrin-B1 to ephrin-B3, in the target regions of these neurons. In situ hybridization studies showed that only ephrin-B2 was ex- pressed at high levels in the striatum. The highest levels of expression were detected in the nucleus accumbens and olfactory tubercle (the ventromedial striatum) (see Figs. 1D,E, 2). Only very low levels were found in the caudate putamen. Thus, ephrin-B2 mRNA was differentially expressed in the striatum. Ephrin-B2 expression in the ventromedial striatum was detected Figure 3. Reduction of EphB1 expression in the substantia nigra by at low levels at E18 to P1 and reached the highest level at 6-hydroxydopamine. A, Reduction of the levels of dopamine and the approximately P7 (Table 2). Low levels of expression persisted in metabolites (DOPAC and HVA) in the striatum of 6-hydroxydopamine- lesioned mice. The data collected are from two animals for each treat- adult (Table 2). These observations indicate that ephrin-B2 ex- ment. B, Reduction of EphB1 hybridization signals in the pression is complementary to that of EphB1, suggesting that the 6-hydroxydopamine-lesioned substantia nigra. The experiments were re- ligand–receptor interaction may restrict the substantia nigra do- peated three times, and two animals were used in each experiment. *p Ͻ paminergic neurons from projecting to the ligand-rich areas, such 0.02; ANOVA. as the nucleus accumbens and olfactory tubercle. Ephrin-B2 selectively inhibits neurite outgrowth of the Dopaminergic neurons were detected with TH immunocyto- substantia nigra, but not the ventral tegmental area, chemical staining after2dinculture. These studies showed that dopaminergic neurons the ventral tegmental dopaminergic neurons grew long neurites The hypothesis that negative interaction between EphB1 and on both the control and the ephrin-B2-expressing cells (Fig. ephrin-B2 serves to restrict substantia nigra dopaminergic neu- 4A,B). However, neurite outgrowth from substantia nigra dopa- rons from projecting to nucleus accumbens is consistent with minergic neurons was reduced significantly on ephrin-B2- known functions of the Eph ligands. This hypothesis predicts that expressing cells (Fig. 4C), although these neurons grew well on axonal growth from substantia nigra, but not from the ventral the control cells (Fig. 4D). The average neuritic length of the tegmental area, is inhibited by ephrin-B2. To test this prediction, substantia nigra neurons on ephrin-B2 cells was only 50% of that we examined the effect of ephrin-B2 on dopaminergic neurite on control cells (Fig. 5A). The inhibition was observed through- outgrowth with a coculture assay. In this assay, ephrin-B2 was out the length distribution; for example, Ͼ70% of substantia nigra stably expressed on the surface of NIH-3T3 cells, because the axons on control cells were longer than 100 ␮m. In contrast, only biologically active ligands require membrane anchorage. Dopa- ϳ19% of the substantia nigra axons were longer than 100 ␮m minergic neurons from the substantia nigra and ventral tegmental when they were cocultured with ephrin-B2-expressing cells (data area of E18 rat embryonic brain were dissected and overlaid on a not shown). No significant inhibition on ventral tegmental dopa- confluent monolayer of control or ephrin-B2-expressing cells. minergic neurite outgrowth was observed (Fig. 5A). 2094 J. Neurosci., March 15, 1999, 19(6):2090–2101 Yue et al. • Specification of Dopaminergic Pathways by Ephrins

Table 2. Developmental expression of EphB1 and ephrin-B2 in the ascending midbrain DA systems

EphB1 in the midbrain Ephrin-B2 in the striatum Stages SN VTA NA OT MCP LCP E18 ϩϩϩ ϩ Ϯ Ϫ Ϯ Ϫ P1 ϩϩϩ ϩ ϩ ϩ Ϯ Ϫ P3 ϩϩϩ ϩ ϩϩ ϩ ϩ Ϫ P7 ϩϩϩ Ϯ ϩϩϩ ϩϩ ϩϩ ϩ Adult ϩϪϩϩϩϩϮ

E, Embryonic stage; P, postnatal stage; DA, dopamine; SN, substantia nigra; NA, nucleus accumbens; OT, olfactory tubercle; MCP, medial caudate/putamen; LCP, lateral caudate putamen.

Figure 4. Ephrin-B2 inhibits the neurite outgrowth of rat E18 substantia nigra neurons. Medial (ventral tegmental area) or lateral (substantia nigra) mesencephalic dopaminergic neurons were dissected and cocultured with a confluent monolayer of ephrin-B2-expressing cells for 48 hr. Dopaminergic neurons were detected by immunocytochemical staining with an anti-TH antibody. A, B, Ventral tegmental dopaminergic neurons cocultured with ephrin-B2-expressing or control cells, respectively. C, D, Substantia nigra DA neurons cultured on the ephrin-B2-expressing or control cells, respectively. E, F, Substantia nigra dopaminergic neurons cultured on ephrin-B2-expressing or control cells in the presence of 6 ␮g/ml of ephrin-B2-Fc. Dopaminergic neurons are stained darkly. Ephrin-B2-expressing or control NIH-3T3 cells appear as faintly stained background. Scale bar, 30 ␮m. Yueetal.• Specification of Dopaminergic Pathways by Ephrins J. Neurosci., March 15, 1999, 19(6):2090–2101 2095

that the inhibition is specific. Thus, the interaction between EphB1 and ephrin-B2 resulted in selective inhibition of neurite outgrowth from the substantia nigra dopaminergic neurons, which express high levels of EphB1, but had no effect on the ventral tegmental area neurons, which express only low levels of the receptor. Induction of cell loss of the substantia nigra dopaminergic neurons by ephrin-B2 In addition to the inhibition of neurite outgrowth of the substan- tia nigra neurons, ephrin-B2 induced a significant decrease of the substantia nigra neuron number in the coculture (Fig. 6A). In the presence of ephrin-B2 the number of the tyrosine hydroxylase- positive substantia nigra neurons was only 41% of that in the control. However, there was no significant reduction in the num- ber of ventral tegmental dopaminergic neurons when they were cocultured with ephrin-B2-expressing cells, as compared with control cells (Fig. 6A). The reduction in dopaminergic neuron number on ephrin-B2-expressing cells was not attributable to a decrease in adhesion, because the number was similar to the control after 12 hr of coculture (Fig. 6B). However, significant neuron loss was apparent by 48 hr of culture. By 72 hr, only 57% dopaminergic neurons survived, in comparison with an 80% survival rate on control cells (Fig. 6B). Furthermore, soluble ephrin-B2 increased the substantia nigra dopaminergic neuron number in a dose-dependent manner (Fig. 6C). To examine whether the decrease in cell number was attributable to apopto- sis, we added methylated Boc-Asp-FMK (BAF; Enzyme System Products, Livermore, CA), a general caspase inhibitor, to the culture medium. BAF significantly increased both the survival rate and the neuritic length of the substantia nigra dopaminergic neurons (Fig. 7). These observations suggest that ephrin-B2 not only inhibits the growth of neurites from the substantia nigra dopaminergic neurons but also induces the loss of these neurons. Induction of ephrin-B2 expression in the striatum by addictive drugs Our observations indicate that ephrin-B2 and EphB1 may regu- late the development of the ascending midbrain dopaminergic pathways by negatively regulating neurite outgrowth and survival of the EphB1-positive neurons in the substantia nigra. To exam- Figure 5. Quantitative analysis of ephrin-B2 effects on the mesence- ine potential roles in plasticity of the dopaminergic systems, we phalic dopaminergic neuritic outgrowth. A, Average neuritic length of the ventral tegmental (VTA) and substantia nigra (SN) dopaminergic neurons analyzed the effects of acute treatment of drugs of addiction. cocultured with ephrin-B2-expressing or control cells. The data shown are Subcutaneous injection of a total of 120 mg/kg cocaine or Ϯ the averages of neuritic length ( SEM). The number of neurites quanti- D-amphetamine dramatically induced ephrin-B2 expression in the tated is indicated above each column. Data were collected from six striatum (Fig. 8). Ephrin-B2 induction by cocaine was dose- independent experiments. The difference in the neuritic length of sub- stantia nigra neurons between the control and ephrin-B2-expressing cells dependent, reaching a saturation concentration at 120 mg/kg is significant (Student’s t test; p Ͻ 0.005). B, Soluble ephrin-B2 reduces the (total dose) (Fig. 9A). Quantitative analyses indicated that the inhibitory effect on substantia nigra DA neurite outgrowth by ephrin-B2- induction appeared to be stronger in the nucleus accumbens, expressing cells. Dopaminergic neurons were cocultured with the ephrin- moderate in medial caudate putamen, and lowest in lateral cau- B2-expressing cells in the presence of various concentrations of the date putamen (see Fig. 8D). The induction was evident by 6 hr soluble ligand as indicated. Neuritic length was measured and tabulated as in A. The data presented are from four independent experiments. Note and reached the highest level by 24 hr after injection (Fig. 9B). that the soluble ligand increases the length of DA neurites in a dose- The level of ephrin-B2 decreased slowly as compared with that of dependent manner. *p Ͻ 0.001; ANOVA. the control over a period of7dafter the first 24 hr. Similar results have been obtained with mice treated with amphetamine (see Fig. It has been shown previously that soluble Eph ligands are 8D). The ability of addictive drugs such as cocaine and amphet- antagonists for native membrane-bound ligands (Gao et al., amine to induce the expression of ephrin-B2 indicates that the 1996). To examine the specificity of inhibition, we added soluble Eph family ligand also may play a role in plasticity of the adult ephrin-B2 to the coculture. The presence of soluble ligand re- midbrain dopaminergic pathways. duced the inhibitive effects of ephrin-B2 on neurite outgrowth in a dose-dependent manner (Figs. 4E,5B). The presence of 6.0 DISCUSSION ␮g/ml soluble ephrin-B2 in large part reversed the inhibition on The current study has examined the roles of a specific Eph ligand the substantia nigra neurite outgrowth by ephrin-B2, indicating and receptor in regulating the axonal growth of developing do- 2096 J. Neurosci., March 15, 1999, 19(6):2090–2101 Yue et al. • Specification of Dopaminergic Pathways by Ephrins

paminergic neurons from the ventral mesencephalon. Our results show that EphB1 and ephrin-B2 are transcribed in mutually exclusive patterns in different dopaminergic neuronal populations and their targets, constituting distinct neural pathways. Further- more, ephrin-B2 specifically inhibits axonal growth and induces cell loss of the receptor-rich substantia nigra dopaminergic neu- rons while having no negative effects on the ventral tegmental area neurons. These observations indicate that ephrin-B2 may function to prevent the substantia nigra dopaminergic neurons from innervating the ligand-rich ventromedial striatum by inhib- itory interactions, thus contributing to the specification of distinct neural pathways. We further report that addictive drugs, cocaine and amphetamine, induce ephrin-B2 expression in adult striatum. These observations together suggest that the Eph family ligand and receptor may participate both in the elaboration of dopami- nergic circuits during development and in drug-induced plasticity in adult.

EphB1 and ephrin-B2 expression and the ascending dopaminergic pathways A large body of evidence indicates that the mesolimbic pathway plays a key role in the motivational aspects of drug addiction as well as emotions and goal-oriented behavior in general (Koob, 1992; Self and Nestler, 1995; Hyman, 1996). In contrast, the appears to be essential for motor functions (Di Chiara et al., 1992), the degeneration of which is a hallmark of Parkinson’s disease. The correlation of the topographic distri- bution of dopaminergic terminals in the striatum and the functional differentiation of the psychological and motor func- tions suggests that the topographic arrangement is critical to the functions of the midbrain dopaminergic systems. Although it has been known for nearly 30 years that the ascending midbrain dopaminergic neurons project to their striatal targets in a topographic manner, the signals regulating the pro- jection have not been identified. In this study we show that EphB1, a receptor of the Eph family, is expressed at high levels in the substantia nigra but at low levels in the ventral tegmental area. Complementary to EphB1 expression, ephrin-B2, a ligand of the receptor (Bergemann et al., 1995; Cerretti et al., 1995; Brambilla et al., 1996), is transcribed at high levels in the ventromedial region of the striatum, including the nucleus accumbens and olfactory tubercle, but at low levels in the dorsolateral striatum. Thus, substantia nigra dopaminergic neurons expressing high levels of the receptor EphB1 project to the dorsolateral striatum, which expresses low levels of the ligand ephrin-B2. Conversely, ventral tegmental neurons, which express low levels of the recep- tor, project to the ventromedial striatum, including the nucleus accumbens and the olfactory tubercle, where high levels of ephrin-B2 are transcribed (Fig. 10). The complementary expres- sion of receptor and ligand in presynaptic and postsynaptic fields is reminiscent of the complementary expression of other Eph Figure 6. Selective induction of the cell death of the substantia nigra receptors and ligands in retinotectal system and hippocamposep- dopaminergic neurons by ephrin-B2. Ventral tegmental (VTA) or sub- tal system and is consistent with their known roles as negative stantia nigra (SN) mesencephalic neurons were dissected and cocultured with control or ephrin-B2-expressing cells. After various times of cocul- guidance cues in topographic map formation (for review, see ture the cells were fixed and stained with anti-TH antibody to identify the dopaminergic neurons. A, Ephrin-B2 decreases the survival of the sub- stantia nigra dopaminergic neurons. The cocultures were maintained for 4 48 hr in this set of six experiments. The difference in the substantia nigra neuron number between control and ephrin-B2 cultures is significant; of ephrin-B2. Neurons were cocultured for 48 hr. The data presented *p Ͻ 0.01; Student’s t test. B, Time course of neuronal loss. The differ- represent the average of four different experiments (ϮSEM). The differ- ences in the neuron number between control and ephrin-B2 cultures at 48 ences among the number of TH-positive neurons cocultured with ephrin- and 72 hr are significant (three experiments; *p Ͻ 0.05; Student’s t test). B2-expressing cells in the presence of 0, 3, or 6 ␮g/ml soluble ephrin-B2 C, Inhibition of cell death by a specific ligand antagonist: the soluble form are significant. *p ϭ 0.001; ANOVA. Yueetal.• Specification of Dopaminergic Pathways by Ephrins J. Neurosci., March 15, 1999, 19(6):2090–2101 2097

Pasquale 1997; Flanagan and Vanderhaeghen, 1998; Zhou, 1998) (also see Cheng et al., 1995; Drescher et al., 1995; Nakamoto et al., 1996; Monschau et al., 1997; Frise´n et al., 1998).

Ephrin-B2 regulates neurite outgrowth and cell survival of dopaminergic neurons The prediction of growth-inhibiting effects of ephrin-B2 on sub- stantia nigra neurons that are EphB1-positive is supported by in vitro assays in this study. Neurite outgrowth of substantia nigra dopaminergic neurons is inhibited by ephrin-B2. In contrast, the growth of ventral tegmental dopaminergic neurons, which nor- mally project to the ligand-rich nucleus accumbens and olfactory tubercle, is not affected. These observations provide further support to the notion that the ligands of the Eph family establish inhibitory domains in the nervous system to prevent inappropri- ate innervation (Drescher et al., 1995; Gao et al., 1996, 1998; Nakamoto et al., 1996; Monschau et al., 1997; Frise´n et al., 1998). Interestingly, in the coculture assay, ephrin-B2 also induces a significant loss of dopaminergic neurons from the substantia nigra, but not from the ventral tegmental area. There are at least two possible explanations for the cell loss. Reduction of cell number could be attributable to a decrease in adhesion because of the reduction in neuritic length by ephrin-B2. Alternatively, ephrin-B2 induces the death of substantia nigra dopaminergic neurons. We consider the first explanation unlikely for the fol- lowing reasons: (1) at 12 hr of culture the number of neurons on ephrin-B2-expressing and control cells is comparable, although neurites are very short at this time; (2) in our previous study (Gao et al., 1996), hippocampal neurons lacking neurites (because of inhibition by ephrin-A2) do not detach from underlying cells; (3) ventral neurons undergo apoptotic cell death in the presence of ephrin-A5, as revealed by both cell counts and TUNEL labeling (Y. Yue and R. Zhou, unpublished data). Thus, the reduction of the number of substantia nigra dopaminergic neurons is likely attributable to cell death induced by ephrin-B2. Although much remains to be done to establish a role of ephrin-B2 in inducing apoptotic cell death, the fact that Boc-Asp- FMK (BAF), a general apoptosis inhibitor, increases cell survival is consistent with this proposal. Furthermore, the anti-apoptotic gene bcl-2 has been shown to promote axon regeneration (Chen et al., 1997), suggesting that molecules regulating cell death may regulate axonal growth also. Earlier studies of the retinotectal development demonstrate that the maturation of the retinotectal map is accompanied by the death of retinal ganglia cells (Rager and Rager, 1976, 1978; Hughes and McLoon, 1979; McLoon and Lund, 1982; Jenkins and Straznicky, 1986). Although it has been proposed that an insufficient number of trophic factors present in the targets or the Figure 7. An apoptosis inhibitor, Boc-Asp-FMK (BAF), reduces the inability to form appropriate synaptic connections may be respon- inhibitory effects of ephrin-B2. Substantia nigra DA neurons were cocul- sible for ganglion cell death (Hughes and McLoon, 1979), our tured with ephrin-B2-expressing cells or control NIH-3T3 cells in the presence of various concentrations of BAF. After 48 hr of coculture observations raise the possibility that the interaction between the the DA neurons were detected with anti-TH antibody and quantitated Eph ligands and receptors also may play a role in eliminating for the number of surviving neurons and the lengths of neurites. A,BAF mistargeted cells during the development of topographic maps. increases the number of TH-positive neurons. B, BAF increases the average neuritic length. The number of neurites quantitated is indicated The timing of expression of the ligand–receptor pair is consis- above each column. C, Neuritic length distribution in the presence of tent with a function in establishing target specificity of dopami- BAF. The distribution was plotted with data from B.TheControl repre- nergic neurons. Dopaminergic innervation occurs primarily in the sents the neurite length distribution of substantia nigra DA neurons ␮ first 4 postnatal weeks (Loizou, 1969, 1972; Coyle and Axelrod, grown on control-transfected NIH-3T3 cells. The absence of BAF (0 M) on ephrin-B2-transfected cells represents the condition in which the most 1972; Olson et al., 1972; Seiger and Olson, 1973). High differential potent inhibition of neurite outgrowth was obtained. The data presented expression of both the ligand and the receptor was observed in the are the average of three separate experiments. *p Ͻ 0.001; ANOVA. 2098 J. Neurosci., March 15, 1999, 19(6):2090–2101 Yue et al. • Specification of Dopaminergic Pathways by Ephrins

Figure 8. Induction of ephrin-B2 in the striatum by cocaine and amphetamine. Adult mice were injected subcutaneously with a total dose of 120 mg/kg cocaine or D-amphetamine. The mice were killed 24 hr later and analyzed for ephrin-B2 expression with in situ hybridization. Four striatal areas, the nucleus accumbens (NA), medial caudate putamen (MCP), lateral (LCP) caudate putamen, and the olfactory tubercle (OT), were quantitated. A, B, Bright- and dark-field photomicrographs, respectively, of a control (saline-injected) adult mouse brain section through the striatum hybridized with an antisense ephrin-B2 probe. C, Dark-field photomicrograph of an ephrin-B2-hybridized section through the striatum of a cocaine-treated (24 hr after treatment at 120 mg/kg total dose) adult mouse brain. D, Quantitative analysis of ephrin-B2 induction by cocaine and amphetamine. The hybridization signals were measured for the percentage of the area covered by the silver grains. The data presented represent the average of three different animals in each experimental group. The areas quantitated are indicated in Figure 1D. The differences in signal density between control and drug-treated animals are significant. *p Ͻ 0.001; **p Ͻ 0.05; ANOVA. ac, Anterior commissure; cc: corpus callosum; Ctx, cerebral cortex; LV, lateral ventricle. Scale bar, 0.62 mm.

early postnatal period, and peak ligand expression was found in Role of ephrin-B2 in drug-induced plasticity the P7 nucleus accumbens (Table 2). Thus, ephrin-B2 and EphB1 Our observations that ephrin-B2 is induced by acute administra- may participate in the specification of distinct dopaminergic path- tion of cocaine and amphetamine suggest that the Eph ligand may ways by negatively regulating axonal growth and cell survival of play a role in drug-induced plasticity. Addictive drugs such as substantia nigra dopaminergic neurons. cocaine, amphetamine, and heroin exert their rewarding effects of Yueetal.• Specification of Dopaminergic Pathways by Ephrins J. Neurosci., March 15, 1999, 19(6):2090–2101 2099

Figure 10. Schematic representation of the relationship between EphB1– ephrin-B2 expression and the topographic projection pattern of midbrain dopaminergic neurons to the striatal targets. In the central dopaminergic system EphB1 was detected at high levels (shown in red) in the substantia nigra (SN). In contrast, little expression was observed in the ventral tegmental system (VTA), which also contains large numbers of dopami- nergic neurons. Complementary to EphB1 expression, its ligand, ephrin- B2, is expressed in the developing striatum at high levels (shown in blue) in the nucleus accumbens and olfactory tubercle, the ventromedial regions of the striatum, with only low levels of expression in the dorsolateral region, the caudate putamen (CP). We hypothesized that the interaction between EphB1 and ephrin-B2 may result in an inhibitive signal (indicat- ed by an X) to restrict substantia nigra axons from innervating the ventromedial striatum, thus contributing to the establishment of the mesostriatal and mesolimbic pathways.

Drug addiction is thought to reflect plastic changes in the neural circuitry mediating the effect of the drug (Nestler et al., Figure 9. Dose–response and time course of ephrin-B2 induction by cocaine. A, Dose–response of ephrin-B2 induction. Different doses of 1993; Hyman, 1996). There are at least two basic mechanisms cocaine (30–180 mg/kg) were injected subcutaneously (in four divided underlying plasticity in general. The first is biochemical changes doses). At 24 hr after injection the mice were killed and analyzed for in neurons and related circuits that, in turn, may lead to electro- ephrin-B2 expression, as in Figure 8. The nucleus accumbens showed the physiological changes. For instance, upregulation of the cyclic highest level of induction. B, Time course of ephrin-B2 induction. Mice treated with 120 mg/kg cocaine (total dose) were killed at various times, AMP pathway has been considered a key event in adaptation to as indicated, for analysis of ephrin-B2 expression as in A. The data for opiates in locus ceruleus and to cocaine in the nucleus accumbens each dose or time point represent the average of three different animals in (Nestler et al., 1993; Hyman, 1996). The second is structural the same treatment group. Animals for each experiment were examined changes of the synapses, neurons, or pathways involved. It has in the same in situ analysis with the same riboprobe. LCP, Lateral caudate putamen; MCP, medial caudate putamen; NA, nucleus accumbens; OT, been shown that chronic morphine treatment reduces the size of olfactory tubercle. *p Ͻ 0.001; **p Ͻ 0.005; ANOVA. ventral tegmental dopamine neurons (Sklair-Tavron et al., 1996). Growth of new synapses has been demonstrated in long-term memory, associated with rapid and transient modulations of mol- and in part via interactions with the dopami- ecules such as NCAM-related cell adhesion molecules, which also nergic system. Cocaine, for example, acts by inhibiting the dopa- regulate axonal outgrowth and guidance in the developing ner- mine transporter and has no effects on knock-out mice lacking the vous system (for review, see Bailey and Kandel, 1995). transporter (Giros et al., 1996). It is thought that the reinforcing Whether changes in neuronal connections are induced by drugs efficacy of drugs is derived by a release of dopamine into the of abuse has not been examined. However, our observation that accumbens, a notion supported by studies in which lesions are the Eph family ligand ephrin-B2 is upregulated in the dopamine made or dopaminergic antagonists are injected in the accumbens targets by acute cocaine treatment suggests that modulation of the (Roberts et al., 1980; Dworkin and Smith, 1987; Koob and Goe- topographic projection of the dopamine system may be a novel ders, 1989; Koob, 1992; Robledo et al., 1992). The induction of mechanism of drug-induced plasticity. Consequently, the ephrin- ephrin-B2 by cocaine and amphetamine in the accumbens is B2–EphB1 interaction may play a role in both the development consistent with a role in addiction. and plasticity of the dopaminergic brain reward circuit. 2100 J. Neurosci., March 15, 1999, 19(6):2090–2101 Yue et al. • Specification of Dopaminergic Pathways by Ephrins

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